The binding of antigen to receptors on B lymphocytes transmits a signal to the lymphocyte. This signal along with factors from T cells and accessory cells results in differentiation of the cell to a plasma cell secreting specific antibody. Indirect evidence suggests that the signal associated with binding of antigen and other factors is transmitted by changes in the plasma membrane and intracellular messenger. However, studies of intracellular events associated with antigen induced B cell triggering or antigen specific tolerance thus far have utilized indirect methods. Direct measurement of intracellular changes was impossible due to the small proportion of specific antigen-binding lymphocytes in most lymphocyte preparations. The principal investigator has developed long term cultures of two normal murine B cell lines which are specific to the hapten dinitrophenyl (DNP). Under the appropriate conditions, the binding of antigen (DNP-Ficoll) or tolerogen (DNP-Murine IgG) to the B cell surface receptor specific for DNP results in either antigen driven B cell differentiation or tolerance induction, respectively. This is a proposal to study the intracellular changes which occur with the binding of the hapten to the receptor on two B cell lines, and to compare the changes which occur when the antigenic and tolerance-inducing forms of the hapten are used. We intend to determine changes in intracellular levels of cylic AMP, cyclic GMP, Ca2+ and cyclic AMP-dependent protein kinases, and profiles of intracellular protein phosphorylation. We will also examine phospholipid methylation and phospholipid phosphorylation in the lymphocyte plasma membrane during induction of B cell differentiation and tolerance. Thus, we will more precisely define the biochemical events associated with hapten-receptor binding which eventually lead to B lymphocyte triggering or tolerance.